University of Lille 10 articles published in JoVE Biochemistry Determination of Glucan Chain Length Distribution of Glycogen Using the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) Method Léa Fermont1, Nicolas Szydlowski1,2, Christophe Colleoni1 1CNRS, UMR8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, University of Lille, 2CNRS, USR3290-MSAP-Miniaturisation pour la Synthèse, l’Analyse et la Protéomique, University of Lille In the present protocol, the Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) technique is used to determine the chain length distribution (CLD) and the average chain length (ACL) of glycogen. Neuroscience Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment Quentin Lemaire1, Marie Duhamel1, Antonella Raffo-Romero1, Michel Salzet1, Christophe Lefebvre1 1U1192-Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse (PRISM), Univ. Lille, INSERM The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments. Genetics High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology Béatrice Colin1, Nicolas Rocq2, Benoit Deprez1, Cyril Couturier1 1Institut Pasteur de Lille, Institut National de la Santé et de la Recherche Médicale (Inserm) U1177, Drugs and Molecules for Living Systems, Université de Lille, 2 This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells. Chemistry Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS! Clémence Simon1, Corentin Spriet1, Simon Hawkins1, Cedric Lion1 1UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, CNRS, UMR 8576, Université de Lille BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta. Biochemistry Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins Clément Danis*1,2, Clément Despres*1, Luiza M. Bessa1, Idir Malki1, Hamida Merzougui1, Isabelle Huvent1, Haoling Qi1, Guy Lippens1, François-Xavier Cantrelle1, Robert Schneider1, Xavier Hanoulle1, Caroline Smet-Nocca1, Isabelle Landrieu1 1UMR8576, CNRS, Lille University, 2UMR-S1172, INSERM CNRS, Lille University We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis. Cancer Research A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model Alexia Mopin1, Virginie Driss1, Carine Brinster1,2 1Institut pour la Recherche sur le Cancer de Lille (IRCL), INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC), 2Université de Lille This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Grünwald Giemsa staining. Environment Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils Sylvie A. Quideau1, Anne C.S. McIntosh2, Charlotte E. Norris1, Emily Lloret3, Mathew J.B. Swallow4, Kirsten Hannam5 1Department of Renewable Resources, University of Alberta, 2Department of Science, Augustana Faculty, University of Alberta, 3Laboratoire Génie Civil et géo-Environnement, Université de Lille, 4Department of Earth and Environmental Sciences, Mount Royal University, 5Forest Ecology & Production, Great Lakes Forestry Centre, Natural Resources Canada Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography. Immunology and Infection Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways Emmanuel Faure1, Perrine Bortolotti1, Eric Kipnis1, Karine Faure1, Benoit Guery1 1Recherche translationnelle: relations hôtepathogènes, Université Lille While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity. Immunology and Infection Proteomic Profiling of Macrophages by 2D Electrophoresis Marion Bouvet1, Annie Turkieh1, Adelina E. Acosta-Martin1, Maggy Chwastyniak1, Olivia Beseme1, Philippe Amouyel1, Florence Pinet1 1Inserm UMR744, University Lille Nord de France Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype. Immunology and Infection A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening Christophe. J Queval*1, Ok-Ryul Song*1, Vincent Delorme*1, Raffaella Iantomasi1, Romain Veyron-Churlet1, Nathalie Deboosère1, Valérie Landry1, Alain Baulard1, Priscille Brodin1 1Inserm U1019 - CNRS UMR 8024, Institut Pasteur de Lille, Université de Lille Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host cell using automated confocal microscopy.